1. Field of the Invention
The present invention relates to a color indicator composition for detecting hydrogen peroxide containing a peroxidase, having improved stability during storage and a quantitative analysis film for detecting hydrogen peroxide comprising the composition which enables operation by a dry process. More particularly, the present invention relates to a color indicator composition for detecting hydrogen peroxide having improved stability during storage which comprises incorporating a specific pyrogallol derivative for preventing inactivation of a peroxidase titer into the composition without substantially interfering with or inhibiting quantitative assay and assaying a detectable chemical species formed by the interaction between a peroxidase and a compound capable of forming a dye through oxidative coupling using hydrogen peroxide as a substrate, as well as a quantitative analysis film for detecting hydrogen peroxide comprising such a composition having improved stability during storage, which enables operation by a dry process.
Various quantitative analysis films, particularly multi-layered analysis films which permit colorimetric analysis of hydrogen peroxide by dry process--in which a peroxidase is involved or which is catalyzed by a peroxidase--have been proposed and some of them have been put in practical use. Among them, there are quantitative analysis films for analysis of glucose, uric acid, cholesterol, choline esterase, creatine, etc., in the living body by a dry process which comprises, in sequence, reacting the same with the appropriate oxidizing enzyme or reacting the reaction product formed during an enzyme reaction with the appropriate oxidiziing enzyme, reacting the thus released hydrogen peroxide with a color indicator to form color, and then measuring the formed color. In order to enhance the speed of examination, avoid complicated operation and reduce cost, demands for dry type quantitative analysis films have increased, particularly in the field of clinical examination. Thus, quantitative analysis films of the test paper sheet type of the single, or dual layer or multi-layered quantitative analysis type with high accuracy in analysis have been developed.
In particular, multi-layer composite type quantitative analysis films are disclosed in Japanese Patent Application (OPI) (the term "OPI" as used herein refers to a "published unexamined Japanese patent application") 53888/74 (corresponding to U.S. Pat. No. 3,992,158) which have markedly improved accuracy in analysis as compared to conventional quantitative analysis films of the test paper sheet type having a single or dual layers. For purposes of further improving accuracy in analysis and in accordance with the analyte to be assayed, hydrogen peroxide indicators having high detection sensitivity have also been provided in such multi-layer composite type quantitative analysis forms; examples of indicators for detecting hydrogen peroxide and layer structures for such multi-layer analysis films are disclosed in Japanese Patent Applications Nos. (OPI) 40191/76 (corresponding to U.S. Pat. No. 4,042,335) 131089/78, 29700/79 (corresponding to U.S. Pat. No. 4,166,093), 124499/80, etc.
Indicators for detecting hydrogen peroxide used for these quantitative analysis films by a dry process are based on those for detecting hydrogen peroxide employed in conventional quantitative analysis by a dry process and the principles are the same.
Particularly, in the field of clinical examination, a variety of components in the living body are converted into common intermediates through chemical reactions having high specificity, generally utilizing an enzyme reaction having particularly high specificity, to thereby effect quantitative determination. As such a common intermediate, hydrogen peroxide has been utilized in many measurement methods. Further, many colorimetric methods which are inexpensive and promise high accuracy have been developed based on combinations of hydroperoxidase--where a substrate is hydrogen peroxide--and related reagents.
Catalase or peroxidase (POD) is often used as hydroperoxidase and, in particular, many combinations with indicators for detecting hydrogen peroxide utilizing peroxidation of peroxidase are known. Indicators for detecting hydrogen peroxide are classified into two groups: (a) reducible chromogens and (b) combination of a hydrogen donor (developing agent) and a coupler. The former was proposed by Keston, A. S., et al. (Keston, A. S., Specific Colorimetric Enzymatics, Analytical Reagents for Glucose, Abstracts of Papers, 129th Meeting Am. Chem. Soc. page 31C, April (1956), and the latter by P. Trinder (Ann. Cli. Biochem., 6, 24 (1969) and J. Clin. Pathol., 22, 246 (1969)). Improvements on such reducible chromogens, hydrogen donors or couplers are disclosed in Japanese Patent Publications Nos. 33798/72, 16235/79, 37555/76, 44834/78, 12360/79, 24879/80 and 3394/79, Japanese Patent Applications (OPI) Nos. 86392/77, 26188/78, 50991/74, 11892/75, 40585/78, 110897/80, 25892/79, 20471/80 and 101861/80, Japanese Patent Publication No. 2960/80, etc.
Analytical compositions, test papers and multilayer analysis films utilizing such enzymological methods involve advantages and disadvantages resulting from enzymes which are employed as reagents.
A major reason why a variety of enzyme control complicated phenomena of life perfectly without any error lies in the substrate specificity of an enzyme. By the substrate specificity, an enzyme can lead a desired chemical substance (compound to be tested) selective to its reaction system among a complicated composition. Such a high selectivity is one of extreme advantage that cannot be possessed by ordinary chemical reagents.
On the other hand, however, enzymes also involve disadvantages due to the fact that enzymes are proteins unlike chemical catalysts employed in chemical industries, manufacturing industries, etc., although enzymes catalyze many reactions in vivo under mild conditions. A most serious disadvantage is that inactivation of an enzyme activity is liable to occur due to denaturation of proteins based on thermal denaturation or an irreversible reaction of an active chemical species. That is, an enzyme activity is created by the presence of a delicate active site based on a primary, secondary or high-dimensional structure of a complicated protein; an enzyme is inactivated by occurrence of deformation of a protein in its structure due to a chemical reaction with a reactive chemical species of an external source, physical adsorption or the like. When an enzyme contained in a composition for quantitative assay is inactivated, an expected reaction becomes slow and a function is not effected as originally designed. As a result, an incorrect result on analysis is obtained.
In analytical compositions, test paper, multilayer analysis films, and the like in which enzyme(s) is/are used as a reagent(s), thermal stability is poor; further, properties principally possessed by an enzyme that are liable to be inactivated by a chemically active species determine the efficiency of these analytical compositions so that it is generally difficult to store them over long periods of time. Storability as analytical elements is maintained, on a bare basis, generally by storage under refrigeration, storage under freezing, moisture-proofing storage, etc.